Biophysical characterization of SARS-CoV-2 spike protein - receptor interactions

PROJECT SUMMARY/ABSTRACT The current pandemic of Coronavirus Disease-2019 (COVID-19) has had devastating impacts across the world. In order to enter human host cells, SARS-CoV-2, the virus causing COVID-19, uses its surface spike (S) protein to attach to host cell surface receptors. Besides the best-known receptor, ACE2, a number of cell surface proteins, including CD147, neuropilin-1 (NRP1) and DC-SIGN/L-SIGN, have been reported to bind to S protein and mediate SARS-CoV-2 entry. Consistently, our preliminary studies using single-molecule force spectroscopy show that CD147, NRP1 and L-SIGN can bind to the SARS-CoV-2 S protein with comparable affinities to those of ACE2. Therefore, the possible multiple receptor utilization could, at least partially, explain the broad tissue tropism and systemic complications of SARS-CoV-2 infection. However, it remains puzzling how the S protein can bind to these structurally diverse molecules with high affinity. In addition, our all-atom structural modeling data shows that most of the S protein surface is covered by glycans, and only when the S protein's receptor binding domain (RBD) is in the "up position" can it bind to a receptor without glycan interference. Therefore, we hypothesize that limited regions on the S protein that are not covered by glycans, including the RBD in up position, as well as the S1/S2 junctional region, may be responsible for binding all the receptors. In the proposed work, we will systematically test the hypothesis using combined approaches of single-virus force spectroscopy, all- atom molecular modeling and simulation and pseudovirus internalization/entry assays. Moreover, since SARS- CoV-2 has two entry routes (direct viral-host membrane fusion or endocytic/macropinocytic internalization followed by endosomal entry), the exact entry pathways that these receptors mediate are not yet clear. The proposed research will determine whether every individual interaction is more prone to mediate direct viral-host membrane fusion or viral endocytic/macropinocytic internalization. Two specific aims will be pursued: 1) to characterize S protein interactions with host cell membrane receptors and 2) to determine the structural basis of S protein's broad receptor recognition. The study will elucidate the structural and biophysical mechanisms behind S protein's receptor recognition and utilization. Successful completion of this work will allow us to identify new targets for antiviral therapies to treat the systemic complications of COVID-19.